Method of large scale production of Hepatitis A Virus

ABSTRACT

The present invention provides methods of large scale production of Hepatitis A Virus (HAV) on VERO cells bound to microcarrier. The invention also provides for methods of isolation of HAV from the cell culture supernatant of HAV infected VERO cells.

FIELD OF THE INVENTION

[0001] The present invention is directed to methods of large scaleproduction of Hepatitis A Virus (HAV) on VERO cells bound tomicrocarrier. The invention also provides for methods of isolation ofHAV from the cell culture supernatant of HAV infected VERO cells.

BACKGROUND OF THE INVENTION

[0002] Hepatitis A continues to cause sporadic cases of infection,endemics, occasional deaths and is a public health problem all over theworld. The infection is caused by Hepatitis A Virus (HAV) a member ofthe picornavirus family, a group of small non-enveloped RNA viruses. Thevirus particle is 27-32 nm in diameter and is composed of threepolypeptides cleaved from a single polypeptide precursor molecule. Themature virus is composed of polypeptides VP1, VP2 and VP3. The capsidproteins VP1 and VP3 contain the major antigenic sites and are capableto induce neutralizing antibodies (Lemon et al., 1989, In: Semler et al.eds. Molecular aspects of picornavirus and detection. Washington, D.C.:ASM p 193-208).

[0003] Hepatitis A Virus (HAV) is the only hepatotropic virus which canbe isolated from cell culture, but the virus is usually difficult topropagate, with long incubation periods and no cytopathic effect. Binnet al. (1984. J. Clincal. Microbiol. 20: 28-33) tested several primatecell types for replication of HAV and optimal conditions for isolationand production of large quantities of virus. Serum free production ofHAV was shown in BSC-1 cells, a heterodiploid cell line that until nowhas not been used for preparation of vaccines for use in humans. After21 days of culture in roller flasks, virus antigen could be found in thesupernatant and the cell fraction. Cells maintained in serum free mediumsupported viral growth equal to that of cells maintained in serum. Acandidate HAV vaccine was obtained from cells and supernatant fluid ofinfected BSC-1 cells maintained in serum free medium (Binn et al., 1986.J. Infect. Diseases 153: 749-756). However, Simmonds et al. (1985, Appl.Enviromental Microbiol. 49:749-755) found no significant difference ofHAV production at different concentration of serum between 2% and 15% inthe medium with persistently infected cells BSC-1 or AGMK cells. Virusproduction in primary AGKM cells was twice that in BSC-1 cells, but HAVproduced remained predominantly cell associated and only some virus wasfound in the culture fluid. Nasser et al. (1987, Appl. EnviromentalMicrobiol. 53:2967-2971) reported that about seven times more HAV wasproduced in FRhK-4 cells culture in one-half or less the time that wasrequired for BS-C1 cultures, wherein the ratio of cell-associated HAVversus HAV released BSC-1 cells was calculated to be 80% to 20%,respectively.

[0004] Flehmig et al. (1987. J. Medical Virol. 22:7-16) prepared HAVfrom cell culture supernatant of persistently infected normal humanembryonic fibroblasts grown in serum containing medium. Using thesemethods, large amounts of supernatants were produced in NUNC cellfactories and HAV antigen isolated from the supernatant and purified inmultiple steps was used for vaccination tests.

[0005] Even though several primate cell types have been reported tosupport replication of HAV, such as fetal rhesus monkey kidney cell line(FRhk-4), primary African green monkey kidney cells (AGKM), continuousAfrican green monkey kidney cells (BCS-1), these cells are generally notused for human vaccine because it is known that monkey kidneys oftenhave high content of latent simian viruses. Other cell lines cannot beused because of the tumorigenic nature of these cells. Mass productionof primary human epithelial, fibroblast or kidney cells or cell strainsto propagate HAV is also limited by the low passage number of thesecells in culture. In fact, the applicable guidelines of the World Healthorganization (WHO) indicate that only a few cell lines are allowed forvirus vaccine production.

[0006] One of the cell lines which is currently accepted and validatedfor the production of vaccine applicable to humans is VERO cells. VEROcells are a continuous monkey kidney cell line that has been licensedfor use in the manufacture of human vaccines and is currently used forthe production of poliomyelitis and rabies vaccine. Attempts have alsobeen made to use VERO cells for HAV production, but it has been foundthat replication of HAV on VERO cells is limited because VERO has atemperature restriction of viral growth. In addition, virus is neverfound in the supernatant fluids of infected cells. (Locarnini et al.,1981, J. Virol. 37:216-225). U.S. Pat. No. 4,783,407 discloses theproduction of HAV on VERO cells in roller bottles at a temperature nohigher than 33° C. to overcome the temperature restriction. HAV antigenwas obtained by freeze-thawing of the cultured cells and release ofintracellular produced virus. A commercial vaccine based on propagationof HAV on VERO cells has never been described.

[0007] So far, formalin inactivated HAV vaccines have been produced forclinical trials (Andre et al., 1990, In: Melnick (ed): Prog. Med. Virol.Basel, Karger 37:72-95, Armstrong et al, 1993, J. Hepatology 18:20-26)and two are commercial available, which induce long-lasting immunity andprotection from primary infection. The manufacturing process of thecurrently available inactivated HAV whole virus vaccines uses the humanembryonic lung fibroblast cell line MRC-5 as host cells in Nunc CellFactories (NCF), wherein the HAV antigen used for vaccine production isobtained form the cell lysate of intracellularly produced virus, becauseHAV antigen is not efficiently released into the culture supernatant andmethods to concentrate the large volume are costly (Bishop et al., 1994.J. Virol. Meth. 47:203-216). HAV large scale preparations from the celllysates and the cell culture supernatants contain mixed populations ofvirions and provirions (Bishop et al., 1997. Arch. Virol. 142:2147-2160)and the commercial available vaccine comprises complete mature virionsand empty provirion particles (Andre 1990 supra, Armstrong 1993 supra).Moreover, MRC-5 cells grow slowly in tissue culture and require fetalcalf serum.

[0008] The problems arising from the use of serum in the cell cultureand/or protein additives derived from an animal or human source (e.g.,the varying quality and composition of different batches and the risk ofcontamination with mycoplasma, viruses or BSE-agents) are well known. Ingeneral, serum or serum derived substances like albumin, transferrin orinsulin may contain unwanted agents that can contaminate the culturesand the biological products derived from them. Furthermore, human serumderived additives have to be tested for all known viruses, likehepatitis or HIV, which can be transmitted by serum. Bovine serum andproducts derived therefrom, for example trypsin, bear the risk ofBSE-contamination. In addition, all serum derived products can becontaminated by unknown agents. Therefore, many attempts are being madeto provide efficient host systems and cultivation conditions that do notrequire serum or other serum derived compounds.

[0009] The production process is as important as the medium. The onlyprocess which is economically feasible is a reactor process because thescale-up can be made appropriate to the market size and the vaccinedoses needed. For adherent cells the carrier process with a classicalmicrocarrier is currently the best choice for large scale cultivation ofthe cells needed for virus propagation. Current processes based onmicrocarrier culture allow production of viral antigen using fermentersizes of up to several thousand liters.

[0010] Widell et al.(1984, J. Virol. Methods 8:63-71) used microcarriercell culture systems of FRhk-4 cells for large scale production of HAVand found intra-and extracellular virus. Virus production per cell usingthe microcarrier system was similar to a conventional culture grown inflask. On the other side, Junker et al (1992, Cytotechnol. 9:173-187)showed that HAV infected MRC-5 cells bound to conventional Cytodexmicrocarriers only yielded 30% HAV antigen compared to cells grown inflasks because of the tendency of MRC-5 cells to form microcarrier andcell aggregates. WO 95/24468 discloses MRC5 cells grown on aggregatedglass-coated microcarriers for HAV production in a perfusion system,wherein the bulk of virus is found in the cells. In the systemdescribed, higher concentrations of serum between 2-10% allowed greaterproduction of HAV than at low level concentration of 0.5-2% of serum.However, when Aunins et al. (1997, In: Carrondo et al. (eds), AnimalCell Technology, p.175-183) compared different manufacturingtechnologies such as Nunc Cell Factories (NCF), microcarriers, staticmixed reactors and CellCubes, they found that glass-coated microcarriersas described in WO 95/24468 allowed the formation of stable aggregatesand production of HAV. The monodisperse microcarrier suspensions,however, could not be maintained for the duration of the culture, andproductivity of the glass aggregate microcarrier process wasapproximately half of static culture under similar conditions. Aunins etal. 1997 (supra) concluded that a microcarrier culture of the HAV strainused was not feasible.

[0011] The worldwide market demand for HAV vaccines is in the order of100 Million doses per year. Efficient vaccine production requires thegrowth of large-scale quantities of virus produced in high yields from ahost system. The process and cultivation conditions under which a virusstrain is grown is of great significance with respect to achieving anacceptable high yield of the strain. Thus, in order to maximize theyield of the desired virus, both the system and the cultivationconditions must be adapted specifically to provide an environment thatis advantageous for the production of the desired virus. Therefore, acontinuing need exists for safe and effective methods to produce virusesand antigen. Moreover, there is a need for an approach to viralpropagation, employing materials that are already available andrequiring a minimal number of time-consuming manipulations, wherein theselection of a combination of host cells, culture medium, growthconditions and production system is essential to achieve an efficientproduction process.

SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to provide a method ofproduction of HAV antigen.

[0013] It is another object of the present invention to provide for amethod for production of HAV in serum free or serum and protein freemedium.

[0014] It is another object of the invention to provide for productionof HAV without use of an animal-derived protease during subculture andpassaging of the cell culture.

[0015] It is another object of the invention to provide for isolation ofcomplete HAV particles.

[0016] It is also an object of the invention to provide a serum free orserum and protein free VERO cell culture infected with HAV whichcontinuously produce HAV antigen

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In accordance with these and other objects, the present inventionprovides a method for continuous production of Hepatitis A virus,comprising the steps of providing a serum free cell culture of VEROcells bound to a microcarrier, infecting said serum free cell culture ofVERO cells with HAV, incubating said cell culture infected with HAV topropagate said HAV, whereby HAV is continuously released into the cellculture medium; and harvesting said HAV released into the cell culturemedium.

[0018] According to the method of the invention, VERO cells bound to amicrocarrier are grown under serum free media conditions at atemperature of about 37° C. The cells are grown from the originalampoule of VERO cells to large scale biomass used in a fermenter forlarge scale production in serum free medium. Prior to infection with HAVthe cell culture temperature is reduced to about 34° C. and furthervirus propagation is performed at this temperature.

[0019] The VERO cells can be bound to a spherical or a porousmicrocarrier during cell culture growth. The microcarrier can be amicrocarrier selected from the group of microcarriers based on dextran,collagen, plastic, gelatine and cellulose and others as described inButler (1988. In: Spier & Griffiths, Animal cell Biotechnology3:283-303). For cell culture growth and during virus infection the samemicrocarrier type can be used. Therefore, according to one embodiment ofthe invention the serum free VERO cells are cultured and infected onspherical microcarriers. According to another embodiment of theinvention the serum free VERO cells are cultured and infected on porousmicrocarriers. It is also possible to grow the cells to a biomass on aspherical microcarriers and subculture the cells when they have reachedfinal fermenter biomass and prior to infection on a porous microcarrieror vice versa. According to this aspect of the invention the serum freeVERO cells are cultured on a spherical microcarrier and infected withvirus when the cells are bound to a porous microcarriers. Sphericalmicrocarrier are those selected from the group of smooth surface such asCytodex I®, Cytodex II® and Cytodex III® (all Pharmacia) and porousmicrocarriers such as Cytopore®, Cytoline® (all Pharmacia).

[0020] The VERO cells bound to microcarrier are infected with HAV at amultiplicity of infection (m.o.i.) between about 0.01 and about 5.

[0021] It has been found that under the conditions described above, HAVis continuously released into the cell culture medium supernatant. Thiswas unexpected because prior art using VERO cells as host for HAVdisclosed that HAV could only be found intracellularly and virusproduced had to be obtained from the cells (U.S. Pat. No. 4,783,407).

[0022] The methods of the present invention provide production of HAV,wherein HAV is continuously produced and released into the cell culturesupernatant. In the method of the invention HAV can be produced for atleast 60 days. The prior art does not describe a cell culture systemthat continuously produces HAV over such a long period of time. By usinga microcarrier culture system and cell culture perfusion, the mediumcontaining the virus is continuously removed from the cell culture andfresh culture medium is added and continuously perfused. The methods ofthe invention provide large volumes of culture medium comprising HAVwhich can be harvested and purified from the cell culture supernatant.

[0023] The parameters for optimal cell culture conditions are a pHbetween about 6.5 and about 8.0, a O₂ concentration between about 15%and about 40%, a stirring speed between about 20 and about 70 rpm, and atemperature at 34° C.±0.2° C. or 37° C.±0.2° C. The culture conditionsare preferably kept constant over the complete time period of virusproduction.

[0024] The use of a virus isolate which has been directly obtained froma primary infected cell culture for virus vaccine production bears therisk of contamination by another virus or an unknown agent. Thecontamination of the virus stock and the cell culture can be avoided byusing a virus stock derived from a defined HAV stock.

[0025] Any strain of HAV can be produced according to the method of thepresent invention. According to one embodiment of the invention thecells are infected with an HAV seed virus that is obtained by using afull-length HAV cDNA to in vitro transcribed HAV RNA and infect VEROcells. By using a cDNA encoding for HAV for production of seed virus, adefined, homogenous virus stock is obtained. The HAV used as seed virusand virus stock can be, for example, HAV HM175/7.

[0026] Besides serum or other protein additives used for cellcultivation, the addition of trypsin derived from an animal source bearsthe risk of contaminating the cell culture by unknown agents. Usually,trypsin from an animal source is used during subculture and passaging ofcell cultures to obtain cell biomass. To avoid any contaminationsderived from an unknown agent or source during HAV virus productionprocess, in the method of the present invention, a protease originatedfrom a microbial source is preferably used for production of cellbiomass from the original ampoule.

[0027] According to one aspect of the invention the cell culture usedfor the production of HAV in the present invention is subcultured fromthe original ampoule to working cell bank and passaged by use of amicrobial protease or a trypsin-like activity of a microbial protease.

[0028] According to a preferred embodiment a purified trypsin-likeenzyme of a microbial protease is used. In particular, the trypsin-likeenzyme is Streptomyces griseus trypsin (SGT), a purified fraction ofPronase, is used. The purified SGT is preferably obtained by a method ofaffinity chromatography on benzamidine and elution of purified SGT withan eluting agent comprising about 0.5 to about 1.2 M arginine. It hasbeen found that the SGT purified by this method is very efficient andcan be used with reduced protein load to the medium due to its highspecific activity. SGT purified from Pronase by other methods known inthe art can be used in the method of the invention as well. Such methodsinclude those described by Yokosawa et al. (1976. J. Biochem.79:757-763) or other chromatography methods.

[0029] According to another preferred embodiment of the invention, serumand protein free culture medium is used for cell culture and growth. Byusing only defined sources, such as minimal medium without addition ofserum or proteins as growth additives for cell biomass production andvirus propagation, a safe virus vaccine production process is provided.

[0030] According to another aspect the invention provides for a methodof isolating complete Hepatitis A virus particles, comprising the stepsof providing a serum free cell culture of VERO cells bound to amicrocarrier, infecting said cell culture with HAV, incubating the cellculture infected with HAV to propagate the HAV, whereby HAV iscontinuously released into the cell culture medium; harvesting HAVproduced and released into the cell culture medium, and isolatingcomplete HAV particles from said HAV harvest of the cell culturesupernatant.

[0031] The term “complete HAV particle” means RNA-containing HAVparticles of mature, infectious HAV virion particles which comprisecapsid proteins VP1, VP2 and VP3, and immature provirions which containVP1, VP3 and VP0 precursor polypeptide.

[0032] The complete HAV particles can be isolated by methods well knownin the art, such as filtering, centrifugation, sedimentation orchromatographic methods. Centrifugation can be performed on asucrose-gradient or CsCl-gradient. Prior to centrifugation larger cellfragments can be removed by e.g. filtration.

[0033] According to another aspect, the invention provides for anHAV-infected serum free VERO cell culture bound to a microcarrier,wherein the cells bound to the carrier continuously produce and releaseHAV into the cell culture medium. The HAV-infected cell culture of theinvention can release HAV continuously for at least 60 days.

[0034] According to a preferred aspect of the invention there isprovided an HAV-infected serum and protein free cell culture of VEROcells culture bound to a microcarrier, wherein the cells bound to thecarrier continuously produces and releases HAV antigen into the cellculture medium.

[0035] Having now generally described this invention, the invention willbe understood by reference to the following examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE 1 Propagation of HAV on VERO Host Cell System

[0036] The HAV strain HM175/7 (kindly provided by Robert Purcell,National Institute of Health, Bethesda, Md.) which has initially beenisolated by a clinical specimen and has been serial passaged in primaryAfrican green monkey cells, which led to the attenuation of the virusstrain, is tested for propagation on VERO cell microcarrier culture.

[0037] VERO cells (African Green Monkey, Cercopthecus aethiops, kidney)are used as production cell line. The cells have been obtained from theAmerican Type Cell Culture Collection, Rockville, Md. at a passagenumber 124 under the designation ATCC CCL 81. The cells are adapted togrow in serum-containing, serum-free, or serum- and protein free mediumas described in Kistner et al. (1998. Vaccine 16:960-968) or WO96/15231. For growth in serum free medium a basal DMEM HAM's F12 mediumsupplemented with inorganic salts, amino acids, sodium bicarbonate (2g/l) and yeast or soy bean extract (1-10 g/l) is used. The working cellbank is prepared without the use of any animal derived mediumcomponents.

[0038] One ampoule of a working cells bank (WCB) of VERO cells culturedin DMEM medium mixed with Ham's F12 nutrient mixture in a ratio 1:1 isresuspended in medium containing serum and in serum free mediumsupplemented either with soy bean or yeast extract (0.1 to 10%).Subculture is performed by using purified Streptomyces griseus trypsin(1 μg/ml) to avoid any agent derived from an animal source which couldcomprise any pathogenic causing agent. After subculture in Roux androller bottles 6-8×10⁷ cells/gram microcarrier (Cytodex III®, Pharmacia)are inoculated in a 12 l stirred tank fermenter. The cells are grown at37° C. for 6-8 days. The culture conditions of oxygen saturation20%+/−10%, pH7.1+/−0.2 are kept constant and stirring speed of 30-60rpm. On the second day after inoculation at a cell density of 6×10⁵ to1×10⁶ cells/ml a virus suspension of HAV HM175/7 with a multiplicity ofinfection (m.o.i.) between 0.1 and 1.0 is pumped into the fermenter at atemperature of either 34° C. or 37° C. After two hours to allow forvirus adsorption, medium perfusion is started. Half of the fermentervolume is exchanged against fresh medium every day. The microcarrier andthe attached cells are retained in the fermenter by a sieve. During thefermentation process pH 7.1, O₂ (30%), stirring speed (30-60 rpm) andtemperature of 34° C. or 37° C. are controlled.

[0039]FIG. 1A shows the HAV produced on VERO cells at 34° C. inserum-free medium and serum-containing (FCS) medium. FIG. 1B shows theHAV produced on VERO cells at 37° C. in serum-free medium andserum-containing (FCS) medium. At days 7, 14, 21 and 28 after infectionthe amount of antigen produced is determined in the cell culturesupernatant and in the cell pellet by means of an HAV specific ELISAassay (Mediagnost). The antigen concentration per 10⁷ VERO cells aredetermined in the cell culture supernatant. The ELISA units (EU) arecalculated as the reciprocal value of the highest antigen dilution thatgives a positive reaction in the ELISA assay.

[0040] HAV strain HM175/7 replicate on VERO cells better at lowertemperature of 34° C. than at 37° C., and better in the absence than inpresence of serum (FIGS. 1A and 1B). At 37° C. in serum-containingmedium no viral antigen production can be observed, wherein at 37° C. inserum-free medium (at higher m.o.i.) virus is produced. Followinginfection of serum free VERO cells with HAV m.o.i. 0.1 or 1 increasedamounts of antigen is detected in the supernatant and cell pellet fromthe 3^(rd) rd week after infection at 34° C. (FIG. 1A). In a cellculture grown at 34° C. in serum containing medium viral antigen isdominantly found in the cell pellet, whereas on VERO cells cultured at34° C. in serum free medium viral antigen is continuously released inthe cell culture supernatant, wherein at about 50% of the viral antigenis found in the supernatant of the culture medium.

EXAMPLE 2 Preparation of HAV Virus Stock for Large Scale Production

[0041] Full length cDNA of the genome of attenuated strain HM175/7cloned in the bacterial plasmid pHAV/7 (Cohen et al., 1987, J. Virol.61:3035-3039) is used to prepare full length genomic RNA by in vitrotranscription. Serum free VERO cells at 34° C. are transfected with invitro transcribed HAV RNA to generate virus stocks free of adventitiousagents. After 6 weeks, HAV specific antigen is detected in the lysate ofinfected cells which are used to further propagate HAV on VERO cellsunder serum free conditions. Table 1 shows the antigen and the virustiter produced after serial passages. The infected cells releasedapproximately 50% of the viral antigen in the cell supernatant. Afterthe 4^(th) passage, the virus stock has a titer of 8×10⁷TCID ₅₀/ml.TABLE 1 Antigen and Virus Titer of serial passages of HAV strain HM175/7after transfection of serum free VERO cells Passages after Total Antigen(EU) Total Titer (TCID₅₀) transfection supernatant cell pelletsupernatant cell pellet Passage 1 n.d. positive n.d. n.d. Passage 2 16000 25 600 n.d. n.d. Passage 3 19 200 25 600 5.2 × 10⁸ 4.7 × 10⁸ Passage4 38 400 51 200 1.5 × 10⁹ 8.9 × 10⁸

[0042] The virus stock HM175/7 obtained after serial passages is usedfor large scale production of HAV antigen on microcarrier system.

EXAMPLE 3 Propagation of HAV HM175/7 on VERO Cells in Serum Free Medium

[0043] HAV HM175/7 as obtained according to Example 2 is seriallypassaged in serum free VERO cells at 34° C. On day 7, 14, and 21 afterinfection the infectious titer and the amount of antigen is determined(Table 2). TABLE 2 Propagation of HAV strain HM175/7 on serum free VEROcells at 34° C. Antigen (EU/5 × 10⁷ cells) Titer (TCID₅₀/5 × 10⁷ cells)Passage No. supernatant cell pellet supernatant cell pellet  7 d neg.  1600 1.3 × 10⁷ 1.1. × 10⁷  14 d  3 200 25 600 1.8 × 10⁸ 2.3 × 10⁸ 21 d 25600 51 200 2.1 × 10⁹ 5.1 × 10⁸

[0044] Virus titers of 5×10⁸ and 2×10⁹ per 5×10⁷ cells are obtained inthe cell pellet and the cell culture supernatant, respectively. Thisdemonstrates that viral antigen is persistently released in the cellculture supernatant by the serum free VERO cells. Three weeks postinfection (p.i.) the percentage of the viral antigen in the cell culturesupernatant is about 50%, while approximately 75% of the infectivity islocalized there (Table 2).

EXAMPLE 4 Production of HAV in Serum Free VERO Cells Propagated onMicrocarrier

[0045] A 6 l fermenter comprising 2×10¹⁰ VERO cells grown onmicrocarrier (Cytodex III®, Pharmacia) in serum free medium is infectedwith HAV strain HM175/7 obtained according to Example 2 with an m.o.i.of 0.5. During a long-term fermentation process at 34° C. the amount ofantigen in the cells and in cell culture supernatant is repeatedlydetermined. For determining HAV produced intracellularly, VERO cellsfrom the cell culture are harvested and adjusted to a cell density of2×10⁷ cells/ml in PBS and lysed by three cycles of freeze/thawing. Afterlow speed centrifugation, the infectious titer in the cell debris andthe cell culture supernatant is determined as well as the amount ofantigen by ELISA assay.

[0046] From day 11 after infection onwards increasing amounts of HAVantigen is detected in the cell culture supernatant. The fermentationprocess is continuously performed and samples are taken over a period of35 days (Table 3). At this time the cells are still viable and produceHAV antigen. The supernatant from day 23 to 35 is pooled and the totalamount of HAV antigen produced is calculated to be 2.5×10⁶ ELISA units.TABLE 3 Antigen production of infected VERO microcarrier cell cultureCell pellet/ml Supernatant Days post infection EU/2 × 10⁷ cell EU/ml 180 10 3 80 — 7 160 — 9 320 11 1280 1 14 1280 2 16 1280 8 18 1280 8 212560 16 23 2560 32 25 2560 40 28 5120 64 30 5120 128 32 5120 160 35 5120320

EXAMPLE 5 Estabishing of Large Scale HAV Production Process

[0047] For establishing of large scale fermentation process, differentstrategies for propagation of HAV are investigated.

[0048] Subconfluent VERO cells, propagated under serum free conditions,are seeded on different types of microcarriers of spherical or porousmicrocarrier, such as Cytodex III®, Cytoline® or Cytopore®, all typesbeing suitable for long-term cultivation process.

[0049] Two days after the cells have been seeded on the different typesof microcarriers, VERO cells are infected with HAV m.o.i. of 1.0. Cellpropagation is performed in a 10 l fermenter at 34° C. with continuousperfusion of growth medium free of serum or free of serum and protein.During the cultivation phase the cell culture supernatant is tested forHAV antigen. The data are summarized in Table 4 . TABLE 4 HAV antigenyield (in EU/ml) after propagation of VERO cells on differentmicrocarriers Days after seeding Microcarrier of infection Cytodex 3Cytopore2 Cytoline 2 16 Neg. Neg. Neg. 18 Neg. 10 4 21 2 40 2 23 4 80 825 4 80 16 28 8 80 8 30 8 80 10 32 8 160 20 35 32 160 20 37 128 160 4039 256 160 40 42 160 160 40 44 160 160 40 46 320 160 40 49 320 160 80 51640 160 80 53 640 160 80 56 640 160 160 58 1280 160 80 60 1280 320 16064 1280 160 320 67 640 160 160 67 160 160 70 160 160 72 160 320 74 160160 77 160 80 79 160 80 81 320 160 83 320 80

[0050] The data of Table 4 show that cells bound to a porousmicrocarrier continuously produce HAV antigen over a long-term period ofat least 83 days. Cells seeded on smooth microcarrier produces highervirus antigen titer at the beginning, but the cells showed tendency toaggregate after some time.

EXAMPLE 6 Long-Term Propagation of Microcarrier Bound Serum Free orSerum and Protein Free VERO Cells

[0051] For large scale production of HAV virus VERO cells grown underserum-free or serum- and protein-free culture medium conditions to abiomass of 1×10¹¹ are seeded on a porous microcarrier. Cells areinfected with HAV with an m.o.i. of 0.1. Propagation of infected cellsat 34° C. up to 350 days are performed with permanent perfusion of thecell culture medium. When virus antigen is detected in the medium, thevirus containing supernatant is collected and stored at 4° C. Theharvest of the serum free cell culture supernatant is started at days35-45 after infection. The virus antigen obtained was calculated for anaverage production of vaccine doses from a 100 l fermenter per l ofmedium per 60 days and is summarized in Table 5. TABLE 5 HAV productionon VERO cells and calculation of productivity for 100 I Scale Lot run #1 2 3 Mean value Mean titer (EU/ml) 640 978 461 693 Volumetricproduction rate 160.000 276.000 128.000 188.00 (EU/I/day) No. ofdoses/I/day (gross) 160 276 128 188 No. of doses/I/day (net) 32 55 26 38No. of doses in 100 days (net) 320.000 552.000 260.00 380.000

EXAMPLE 7 Purification of HAV Antigen from Cell Culture Supernatant

[0052] The cell culture supernatant collected from the perfusion culturemedium as described in Example 6 comprising HAV antigen is separatedfrom the cellular debris by low speed centrifugation or depth filter,and concentrated by ultrafiltration using a 50 K Omega membrane (cut-off50 000 Da, Filtron). The concentrate is further purified bycentrifugation over a 20%-60% sucrose gradient and fractionated. Eachfraction is tested for HAV antigen by a qualitative ELISA assay(Mediagnost). HAV antigen assembled in two peak fractions. The peakfractions are separately pooled and concentrated by high speedcentrifugation.

[0053] During the process described above, the amount of antigen and theprotein content is determined. The two peak pool fractions are analyzedby Western blot analysis with antibodies specific for HAV polypeptidesVP0, VP1 and VP3 as well as a mixture thereof. The peak pool fractions12-19 consist of mature virions (because of the presence of the capsidprotein VP2 and the absence of VP0). The peak pool fractions 22-25contain provirions and/or preprovirions.

[0054] This shows that by the process described HAV is continuouslyreleased in cell culture medium by persistently infected VERO cellsgrown in serum free or serum and protein free medium during large scalemanufacturing process.

[0055] The respective fractions 12-19 and 22-25 are collected, the viruspreparation is subjected to virus inactivation method and theinactivated preparation is formulated in a vaccine composition.

EXAMPLE 8 Purification of Streptomyces griseustrypsin from Pronase

[0056] a) Ion Exchange Chromatography

[0057] 30 g of Pronase (Boehringer Ingelheim) was dissolved in Buffer A(0.02 pyridin, pH 5.0) to a final concentration of 40 mg/ml Pronase. 25ml of the solution was subjected to cation exchange chromatography on CMSepharose Cl 6B (Pharmacia) equilibrated with buffer A). The elution wasperformed at room temperature using a linear gradient with buffer A(0.02 M pryridin) and buffer B (0.75M pyridin pH 5.0) with 5 times thecolumn volume.

[0058] Collected fractions were tested for inhibiting properties bymixing samples of the fractions with soy bean inhibitor in a 1:10 ratio(e.g. 1 mg soy bean inhibitor/100 μg protein) followed by achromatographic substrate assay using S2222. The results were expressedas Δ absorbance units per minute (Δ A/min). The fraction having thehighest inhibiting activity to soy bean inhibitor was further analysedby SDS-PAGE and stained with Coomassie.

[0059] The trypsin activity was measured by chromogenic assay usingN-benzoyl-L-arginine ethyl ester (BAEE, in Tris buffer pH 8.0, 20 mMCaCl₂, 25° C.) as substrate and Δ absorbance units per minute isdetermined. As a control reference, porcine trypsin solution (1 mg/ml)with a specific activity of 13×10³ U/mg was used. The specific activitywas defined as the units of trypsin enzyme activity per mg protein. Theresults are summarized in Table 1.

[0060] The chymotrypsin activity was measured by chromogenic assay using3-carboxymethoxypropionyl-L-arginyl-L-propyl-L-tyrosine-p-notroanilinehydrochloride (S-2586, Chromogenix). The results were expressed Δabsorbance units per minute (Δ A/min). TABLE 6 Purification of Pronaseby ion exchange chromatography Streptomyces griseus Pronase Pronaseunpurified Purified fraction Protein (g) 1 0.08 Specific activity U/mg1.6 × 10³ 16.5 × 10³ Recovery U in % 100 70 Stability by SDS-PAGE n.d.Unstable, low molecular weight fragmentation Inhibition by soy beaninhibitor n.d.   90 ± 0.1 (% inhibition) Chymotryspin activity 450 38 (ΔA/min)

[0061] Table 6 shows that the fractions containing a protein havingtrypsin-like activity, as determined by inhibition test with soy beaninhibitor, can be purified by ion exchange chromatography with aspecific activity which is about 10 times higher than of Pronase andwith a recovery of about 70%. However, the protein is unstable and showsnot a single band, but various bands in SDS-PAGE. This is indicative offragmentation and autocleavage of the protein.

[0062] b) Affinity chromatography on immobilized benzamidine

[0063] A Benzamidine Sepharose 6B fast flow (Pharmacia) columnequilibrated with buffer A (50 mM Tris, 0.5 M NaCl pH 7.0) was loadedwith 40 ml of a Pronase solution (75 mg/ml, buffer A). Elution wasperformed with Buffer B (50 mM Tris, 0.5 M NaCl pH 7.0, 10 mMbenzamidine hydrochlorid pH 7.0), buffer C (0.5 M NaCl, 0.6 M arginine,pH 5.5) or buffer D (0.5 M NaCl, 1 M arginine, pH 5.5).

[0064] The fractions collected were tested for inhibiting propertiesusing soy bean inhibitor, as well as trypsin and chymotrypsin activityas described in Example 8 A. The specific activity was determined asunits of enzyme activity per mg protein. TABLE 7 Purification of Pronaseby affinity chromatography on immobilized benzamidine and elution withbenzamidine Strepromyces griseus pronase Pronase unpurified Purifiedfraction Affinity chromatography and elution with benzamidine (Buffer B)Protein (g) 3 0.13 Specific activity U/mg 1.6 × 10³ 19 × 10³ Recovery Uin % 100 60 Stability by SDS-PAGE stable stable Inhibition by soy beaninhibitor n.d. 99.98 ± 0.1% (% inhibition) Chymotryspin activity n.d.0.1 (Δ A/min)

[0065] The results summarized in Table 7 show that by competitiveelution with benzamidine, 60% of purified trypsin-like activity ofPronase was recovered with a specific activity of about 140 U/μgprotein. However, the purified trypsin-like protease containing fractionis preferably further purified and the benzamidine removed prior to usein processes which involve cell culture growth or production ofbiologicals for application in humans. TABLE 8 Purification of Pronaseby affinity chromatography on immobilized benzamidine and elution with0.6 M arginine and 1 M arginine Streptomyces griseus Pronase Pronaseunpurified Purified fraction Affinity chromatography and elution with0.6 M arginine (Buffer C) Protein (g) 3 0.13 Specific activity U/mg 1.6× 10³ 26 × 10³ Recovery U in % n.d. 63 Stability by SDS-PAGE stablestable Inhibition by soy bean inhibitor n.d. 99.89 ± 0.1% (% inhibition)Chymotrypsin activity n.d. <0.1 (Δ A/min) Affinity chromatography andelution with 1 M arginine (Buffer D) Protein (g) 3 0.13 Specificactivity U/mg 1.6 × 10³ 46.5 × 10³ Recovery U in % n.d. 71% Stability bySDS-PAGE stable stable Inhibition by soy bean inhibitor n.d. 99.99 ±0.1% (% inhibition) Chymotrypsin activity n.d. <0.1 (Δ A/min) LAL(EU/1000 U) 88 <4

[0066] As can be seen from results in Table 8, about 63% of the initialtrypsin-like activity of Pronase was recovered when using a buffercomprising 0.6 M arginine, whereas about 71% is recovered with a buffercomprising 1M arginine. The purified SGT eluted with arginine from abenzamidine affinity carrier also had a higher specific activitycompared to SGT obtained by ion exchange chromatography or elution withbenzamidine from a benzamidine carrier. Further, a product of higherpurity and specific activity was obtained when a buffer comprisingincreasing molarity of arginine was used.

[0067] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

What is claimed is:
 1. A method for continuous production of Hepatitis Avirus, comprising the steps of providing a serum free cell culture ofVERO cells bound to a microcarrier, the method comprising infecting saidserum free cell culture of VERO cells with HAV, incubating said serumfree cell culture of VERO cells infected with HAV to propagate said HAV,whereby HAV is continuously released into the cell culture medium; andharvesting said HAV released into the cell culture medium.
 2. The methodaccording to claim 1, wherein said cells are grown at a temperature ofabout 37° C.
 3. The method according to claim 1, wherein saidtemperature is reduced to about 34° C. prior to infection.
 4. The methodof claim 1, wherein the microcarrier is selected from the group ofspherical or porous microcarriers.
 5. The method according to claim 4,wherein the microcarriers comprise dextran, gelatine, collagen, plastic,or cellulose.
 6. The method according to claim 1, wherein the cells areinfected with a seed virus of HAV strain HM175/7.
 7. The methodaccording to claim 1, wherein the cells are infected with HAV at amultiplicity of infection between about 0.01 and about 5.0.
 8. Themethod according to claim 1, wherein the cell culture is subculturedfrom a working cell bank and passaged by use of a microbial protease ora trypsin-like enzyme of a microbial origin.
 9. The method according toclaim 8, wherein said microbial protease is the trypsin-like enzyme ofStreptomyces griseus Pronase.
 10. The method according to claim 1,wherein HAV is continuously produced for at least 60 days.
 11. Themethod according to claim 1, wherein said serum free cell culture ofVERO cells is a serum and protein free cell culture of VERO cells.
 12. Amethod of isolating complete Hepatitis A virus particles, the methodcomprising the steps of providing a serum free cell culture of VEROcells bound to a microcarrier, infecting said cell culture with HAV,incubating said cell culture infected with HAV to propagate said HAV,whereby HAV is continuously released into the cell culture medium;harvesting said HAV released into the cell culture medium; and isolatingcomplete HAV particles from said HAV harvest of the cell culturesupernatant.
 13. The method according to claim 12, wherein said cellsare grown at a temperature of about 37° C. prior to infection.
 14. Themethod according to claim 12, wherein the cell culture temperature isreduced to about 34° C. after infection.
 15. The method of claim 12,wherein the microcarrier is selected from the group of smoothmicrocarriers or porous microcarriers.
 16. The method according to claim15, wherein the microcarriers comprise dextran, collagen, plastic,polyethylene or cellulose.
 17. The method according to claim 12, whereinthe cells are infected with a seed virus of HAV strain HM175/7.
 18. Themethod according to claim 12, wherein the cell culture is subculturedfrom a working cell bank and passaged by use of a microbial protease ora trypsin-like enzyme of a microbial protease.
 19. The method accordingto claim 18, wherein said microbial protease is the purifiedtrypsin-like enzyme of Streptomyces griseus pronase.
 20. The methodaccording to claim 12, wherein HAV is continuously produced for at least60 days.
 21. The method according to claim 12, wherein the complete HAVparticles are isolated by isopycnic centrifugation.
 22. An HAV-infectedserum free cell culture of VERO cells bound to a microcarrier, whereinsaid cells bound to said carrier continuously releases HAV antigen intothe cell culture medium.
 23. An HAV-infected serum and protein free cellculture of VERO cells bound to a microcarrier, wherein said cells boundto said carrier continuously releases HAV antigen into the cell culturemedium.